Characterization of chemically modified steroids for doping control purposes by electrospray ionization tandem mass spectrometry

Abstract

The discovery of the designer steroid tetrahydrogestrinone (THG) in elite athletes' doping control samples in 2003 demonstrated the availability of steroid derivatives prepared solely for doping purposes. Modern mass spectrometers utilizing electrospray ionization and collisionally activated dissociation (CAD) of analytes allow the structural characterization of steroids and their derivatization sites by the elucidation of fragmentation behaviors. A total of 21 steroids comprising either a 4,9,11-triene, a 3-keto-4-ene or a 3-keto-1-ene nucleus were investigated regarding their dissociation pathways, deuterated analogues were synthesized and fragmentation routes were postulated, permitting the identification of steroidal structures and modifications. Compounds based on a 4,9,11-triene steroid with an ethyl residue at C-13 (gestrinone analogues) generate abundant fragment ions at m/z 241 and 199, whereas the substitution of the C-13 ethyl group by a methyl residue (trenbolone analogues) results in a shift of m/z 241 to 227. Substances related to testosterone with a 3-keto-4-ene structure give rise to abundant fragment ions at m/z 109 and 97 whereas steroids with a 3-keto-1-ene nucleus eliminate the A-ring including the carbons C-1-C-4, in addition to C-19 that is proposed to migrate from C-10 to C-1 under CAD conditions.

Collision-induced dissociation pathways of anabolic steroids by electrospray ionization tandem mass spectrometry

“…Small differences in the chemical structures of the steroids, such as an additional double-bond or a methyl group, result in significantly different fragmentation pathways. The fragmentation pathways proposed in this paper allow interpretation of major product ions of other anabolic steroids reported by other researchers in a recent publication [19]. The proposed fragmentation pathways are helpful for characterization of new steroids.…”

“…Based on the RDB value (Table 1), structure for the radical ion of m/z 266 is proposed (Scheme 3v). On the fragmentation route from the transition-state species (Scheme 3k) to the final product ion (Scheme 3v), either the 13-or the 17-ethyl group may eliminate according to the results of Thevis et al from deuterium-labeled THG [19]. Generation of the product ion of m/z 266 via loss of H 2 O molecule and ethyl radical group from the [M ϩ H] ϩ ion is supported by the results from deuteriumlabeled THG [19] and EI spectrum of THG [22].…”

“…Fragmentation pathways are reported for testosterone and testosterone hydroxyl analogs [15], oxosteroid Girard derivatives [16,17], and steroid oximes [18] under ESI conditions. Recently, Thevis et al reported electrospray ionization tandem mass spectrometric characterization of chemically modified steroids including 21 gestrinone and testosterone analogs [19]. In this paper, formation of major product ions of selected anabolic steroids under ESI (ϩ) MS/MS conditions are interpreted and fragmentation pathways are proposed for those steroids of which fragmentation pathways have not been previously reported.…”

Mass spectrometric analysis of androstan-17β-ol-3-one and androstadiene-17β-ol-3-one isomers

“…Starting from the protonated species m, the elimination of the entire D-ring (Ϫ98 u) by means of a retro-Diels-Alder rearrangement is accompanied by the release of ketene (Ϫ42 u) from the A-ring including carbons 2 and 3 yielding the proposed fragment o at m/z 147. The resulting cross-conjugated -electron system is considered the driving force for this dissociation mechanism.°Budzikiewicz°et°al.° [26]°describe°the°elimination of ketene from steroids with a 3-keto-4-ene releasing C-2 and C-3 upon electron impact, and migration of C-19 from C-10 to C-1 as a Wagner-Meerwein rearrangement has been proposed in fragmentation patterns°of°steroids°bearing°a°3-keto-1-ene°structure° [19].…”

“…Detailed studies have also been made employing steroid derivatives such as trimethylsilylated or acetylated compounds [9 -18]. Liquid chromatography combined with mass spectrometers with an atmospheric pressure ionization interface enables the detection and characterization of steroids that complement conventional gas chromatographic-mass spectrometric (GC-MS) assays, especially for compounds that are less suitable for GC-MS owing to thermo-labile properties [19]. A very recent example is the so-called "designer steroid" tetrahydrogestrinone [20] (13-ethyl-17-hydroxy-18,19-dinor-17␣-pregn-4,9,11-trien-3-one, THG) that has been identified in several doping control samples by means of methods based on liquid chromatography (LC) and tandem mass spectrometry (MS/MS).…”